A semi-synthetic organism that stores and retrieves increased genetic information

 描述（由申请人提供）：生物体由其基因组中编码的信息定义，并且自我们所知的生命进化以来，该信息已使用四个字母的遗传字母表编码，通过（d）G与（d）C和（d）A与dT或U的选择性配对成为可能。第三个非天然碱基对（UBP）的产生将对我们理解生命是什么以及它是如何进化的产生深远的影响，并且可以作为半合成生物体（SSO）的基础，SSO是一种活细胞，可以存储自然遗传字母表之外的信息，并以含有非天然氨基酸的蛋白质的形式检索它。这具有改善人类健康的巨大潜力，因为蛋白质现在构成了一类重要的治疗药物，但它们的效用目前受到二十种天然氨基酸有限的物理化学多样性的限制。 自1999年以来，我们的NIH资助的工作一直集中在UBP的发展。我们的策略是基于使用疏水和包装力，而不是沃森-克里克样氢键，以优化UBP的形成，因为我们发现这种力很强，不利于与天然的，更亲水的核苷酸错配。这项工作在2009年达到了重要的里程碑，我们发现了dNaM和d5 SICS之间形成的UBP，并在2014年工程化了E。基于大肠杆菌的SSO，其在其DNA中稳定地含有该UBP。在过去的一年中，我们继续优化SSO，包括编码三角褐指藻核苷酸三磷酸转运蛋白（PtNTT 2）的基因的优化和基因组整合，这使得dNaMTP和d5 SICSTP导入细胞成为可能。随着优化的PtNTT 2现在在组成型启动子的控制下，SSO更健康，并且总是能够进行非天然的三磷酸盐摄取。虽然我们已经发现UBP的复制存在序列偏差，但我们已经通过继续优化UBP以及引入由CRISPR相关蛋白-9核酸酶（Cas9）介导的纠错机制，在消除偏差方面取得了进展。我们还证明了含有UBP的DNA可以在SSO内转录成含有非天然核苷酸的RNA。尽管仍需要继续探索和优化，但已经确定了创造第一种生命形式的主要挑战，这种生命形式稳定地拥有和检索超出自然遗传字母表编码的信息。这些包括复制的优化，以消除观察到的序列偏差，转录的优化，包括mRNA和tRNA的转录，最后，有效翻译的演示，以及克服这些挑战的策略进行了描述。如果成功的话，我们的努力将产生第一种生命形式，它忠实地存储和检索自然遗传字母表编码之外的信息，并将为生产多样化的治疗性蛋白质提供一个通用平台，这可能会彻底改变医学。

 DESCRIPTION (provided by applicant): Organisms are defined by the information encoded in their genomes, and since the evolution of life as we know it, this information has been encoded using a four-letter genetic alphabet, made possible by the selective pairing of (d)G with (d)C and (d)A with dT or U. The creation of a third, unnatural base pair (UBP) would have profound implications for our understanding of what life is and how it may have evolved, and could serve as the foundation of a semi-synthetic organism (SSO), a living cell that stores information beyond that of the natural genetic alphabet and retrieves it in the form of proteins containing unnatural amino acids. This has great potential to improve human health, as proteins now constitute an important class of therapeutics, but their utility is currently restricted by the limted physicochemical diversity of the twenty natural amino acids. Since 1999, our NIH-funded work has focused on the development of a UBP. Our strategy is based on the use of hydrophobic and packing forces, as opposed to Watson-Crick-like hydrogen bonds, to optimize UBP formation, as we have found that such forces are strong and disfavor mispairing with the natural, more hydrophilic nucleotides. This effort reached major milestones in 2009 with our discovery of the UBP formed between dNaM and d5SICS, and in 2014 with our engineering of an E. coli-based SSO that stably harbors this UBP in its DNA. In the past year, we have continued to optimize the SSO, including the optimization and genomic integration of the gene encoding the nucleotide triphosphate transporter from Phaeodactylum tricornutum (PtNTT2), which makes import of dNaMTP and d5SICSTP into the cell possible. With the optimized PtNTT2 now under the control of a constitutive promoter, the SSO is more healthy and always competent for unnatural triphosphate uptake. While we have discovered that replication of the UBP proceeds with a sequence bias, we have already made progress towards eliminating the bias by continuing to optimize the UBP, and by introducing an error-correction mechanism mediated by CRISPR-associated protein-9 nuclease (Cas9). We have also demonstrated that DNA containing the UBP may be transcribed within the SSO into RNA containing unnatural nucleotides. Although continued exploration and optimization is still required, the major challenges of creating the first form of life that stably harbors and retrieves information beyond that encoded by the natural genetic alphabet have been identified. These include the optimization of replication to eliminate the observed sequence bias, the optimization of transcription, including the transcription of mRNAs and tRNAs, and lastly, the demonstration of efficient translation, and strategies toward overcoming these challenges are described. If successful, our efforts will yield the first form of life that faithfully stores and retrieves infomation beyond that encoded by the natural genetic alphabet, and will result in a general platform for the production of diverse, therapeutic proteins that could revolutionize medicine.